Methods and kits for nucleic acid amplification

ABSTRACT

Compositions and methods are provided for amplifying nucleic acid molecules. The nucleic acid molecules can be used in various research and diagnostic applications, such as gene expression studies involving nucleic acid microarrays.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods foramplifying nucleic acid molecules.

BACKGROUND OF THE INVENTION

Microarray technology has become a powerful tool for generating andanalyzing gene expression profiles. Microarray expression analysis,however, generally demands large amounts of RNA that are often notavailable (see Wang et al., BioTechniques 34:394 (2003)). Several RNAamplification techniques have been developed to overcome this problem.These techniques, however, generally suffer from a phenomenon known asamplification bias (see, e.g., U.S. Pat. No. 6,582,906). In these cases,the amplified population of RNA molecules does not proportionallyrepresent the population of RNA molecules existing in the originalsample.

For example, in the method disclosed by Eberwine and colleagues (see,e.g., U.S. Pat. Nos. 5,545,522; 5,716,785; 5,891,636; 5,958,688; and6,291,170), a compound oligonucleotide is utilized for theamplification, wherein the compound oligonucleotide is provided withboth a T7 promoter and a primer. A cDNA copy is created of an initialmRNA transcript using the compound oliognucleotide, with subsequentsecond strand synthesis to create a cDNA that is double stranded. RNAamplification is conducted via the promoter portion of the compoundoligonucleotide, with transcription proceeding off of the cDNA's secondstrand. Since the second strand is used for transcription, the Eberwinemethod produces amplified RNA that is antisense to the initial mRNAsequence (termed cRNA or aRNA).

The Eberwine method, however, introduces a 3′ bias during each of itssteps due to the incomplete processivities (i.e., the inability of anenzyme to remain attached to a nucleic acid molecule) of the enzymesutilized and the positioning of the RNA polymerase promoter (see, e.g.,U.S. Pat. No. 6,582,906 and U.S. Patent Publication No. US2003/0104432).For example, the compound oligonucleotide used to produce first strandcDNA places the promoter at the 5′ end of the cDNA, which corresponds tothe 3′ end of the message. This coupled with the inability of RNApolymerase to complete transcription of some templates (due perhaps tolong polyA tail regions or interference from secondary and tertiarystructures in the template) can result in a 3′ bias in the amplifiedantisense RNA population. In addition, if second strand cDNA synthesisby DNA polymerase is incomplete, these cDNAs will lack functionalpromoters, resulting in a reduced representation of the original RNAmolecule (or possibly a complete absence) in the amplified population.

Applicants' copending patent applications U.S. patent application Ser.Nos. 10/979,052, 11/150,794 and 11/210,602, and InternationalApplication No. PCT/US2004/014325, each specifically incorporated hereinby reference in its entirety, disclose methods for attaching orsynthesizing RNA polymerase promoters onto the 3′ ends of cDNAmolecules. In vitro transcription is initiated by addition of RNApolymerase, resulting in the synthesis of sense RNA (sRNA) moleculeshaving the same orientation as the original RNA molecules from which thecDNA molecules were synthesized. For downstream applications, such asgene expression studies, the sRNA molecules can be reverse transcribedinto cDNA molecules or used in aRNA amplification reactions using theEberwine method described above.

Reverse transcription of the sRNA molecules, however, provides nofurther amplification of the original nucleic acid sequences, limitingits use when small amounts of RNA are involved. Eberwine's aRNA method,while providing amplification, often results in large amounts ofnon-specific artifacts due to the use of a compound oligonucleotidecontaining an intact T7 promoter.

It would be desirable to provide methods and kits for synthesizingantisense RNA (asRNA) molecules directly from sRNA molecules whichprovides increased amplification with low amounts of non-specificartifacts.

SUMMARY OF THE INVENTION

Applicants have invented methods for the synthesis of asRNA moleculesdirectly from sRNA molecules, wherein a partial RNA polymeraserecognition sequence at the 3′ ends of sRNA molecules is converted intoa complete RNA polymerase recognition sequence and ultimately into adouble stranded RNA polymerase promoter. Subsequent RNA transcriptionusing an RNA polymerase that recognizes the double stranded RNApolymerase promoter results in the production of amplified asRNAmolecules. Applicants have discovered that this method of promoterformation and amplification provides lower amounts of non-specificartifacts compared to traditional aRNA amplification methods involvingintact promoter primers.

Accordingly, one aspect of the present invention is directed to a methodfor synthesizing at least one asRNA molecule directly from at least onesRNA molecule, comprising:

-   -   a) providing at least one sRNA molecule having a 5′ end and a 3′        end, said 3′ end comprising a first nucleotide sequence        corresponding to the partial 5′ end of the template strand of a        RNA polymerase recognition sequence;    -   b) annealing to the 3′ end of said sRNA molecule a primer having        a 5′ end and a 3′ end, said primer comprising a second        nucleotide sequence corresponding to at the 5′ end of the        non-template strand of said RNA polymerase recognition sequence        sufficient in length to anneal to said first nucleotide sequence        corresponding to the partial 5′ end of the template strand of        said RNA polymerase recognition sequence;    -   c) extending the 3′ end of said primer such that a double        stranded RNA/DNA duplex is formed;    -   d) degrading at least the 3′ portion of the RNA strand of said        double stranded RNA/DNA duplex, thereby providing at least a        partially single stranded DNA molecule having a single stranded        5′ end, said 5′ end comprising a third nucleotide sequence        corresponding to the complete non-template strand of said RNA        polymerase recognition sequence;    -   e) synthesizing at least a partially double stranded DNA        molecule from said at least partially single stranded DNA        molecule such that said third nucleotide sequence corresponding        to the complete non-template strand of said RNA polymerase        recognition sequence is converted into a double stranded RNA        polymerase promoter; and    -   f) initiating RNA transcription using an RNA polymerase which        recognizes said double stranded RNA polymerase promoter,    -   thereby synthesizing at least one asRNA molecule directly from        at least one sRNA molecule.

In some embodiments, the at least partially double strand stranded DNAmolecule is synthesized by performing second strand DNA synthesis withan exogenous primer. In other embodiments, the at least partially doublestrand stranded DNA molecule is synthesized by extending a remaining 5′portion of the RNA strand of the double stranded RNA/DNA duplex.

As described more fully below, the sRNA molecule can be provided byattaching one or more RNA polymerase promoters to the 3′ end of a cDNAmolecule, followed by one or more rounds of RNA transcription with RNApolymerases which recognizes the RNA polymerase promoters. The cDNAmolecule can be provided by contacting a RNA molecule with a primerhaving a 5′ extension comprising a fourth nucleotide sequencecorresponding to the complement of the first nucleotide sequence in thepresence of a reverse transcriptase. Such reverse transcription primersinclude oligodT primers, random primers or combinations thereof. Uponreverse transcription of the RNA molecule and subsequent RNAtranscription of the resulting cDNA molecule, the fourth nucleotidesequence of the reverse transcription primer becomes the firstnucleotide sequence at the 3′ end of the sRNA molecule.

Applicants have also invented kits for the synthesis of asRNA moleculesdirectly from sRNA molecules, wherein a partial RNA polymeraserecognition sequence at the 3′ ends of sRNA molecules is converted intoa complete RNA polymerase recognition sequence and ultimately into adouble stranded RNA polymerase promoter.

Accordingly, another aspect of the present invention is directed to akit for synthesizing one or more asRNA molecules directly from a sRNAmolecule, comprising: one or more primers comprising a nucleotidesequence corresponding to at least a portion of the 5′ end of thenon-template strand of an RNA polymerase recognition sequence; andinstructional materials for synthesizing asRNA molecules directly fromsRNA molecules using said primer. In some embodiments, the kit furthercomprises reagents and instructional materials for synthesizing sRNAmolecules from which asRNA molecules can be directly synthesized.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and features of the present invention will become apparentfrom the following detailed description considered in connection withthe accompanying drawings. It is to be understood, however, that thedrawings are designed as an illustration only and not as a definition ofthe limits of the invention.

FIGS. 1 a-i is a schematic representation that depicts an embodiment forsynthesis of sRNA molecules according to the methods of the presentinvention;

FIGS. 2 a-d is a schematic representation that depicts an embodiment forsynthesis of asRNA molecules directly from sRNA molecules according tothe methods of the present invention; and

FIG. 3 is a photograph that depicts sRNA and as RNA produced by themethods of the present invention visualized on a 1% agarose gel stainedwith ethidium bromide.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention utilize routine techniques in thefield of molecular biology. Basic texts disclosing general molecularbiology methods include Sambrook et al., Molecular Cloning, A LaboratoryManual (3d ed. 2001) and Ausubel et al., Current Protocols in MolecularBiology (1994).

The present invention relates to methods and kits for amplifying nucleicacid molecules. The terms “nucleic acid molecule”, “RNA molecule”, “sRNAmolecule”, “asRNA molecule”, “aRNA molecule, “cRNA molecule”, “DNAmolecule”, and “cDNA molecule” are each intended to cover a singlemolecule, a plurality of molecules of a single species, and a pluralityof molecules of different species.

The methods of the present invention generally comprise converting apartial RNA polymerase recognition sequence at the 3′ ends of sRNAmolecules into a complete RNA polymerase recognition sequence andultimately into a double stranded RNA polymerase promoter. SubsequentRNA transcription using an RNA polymerase that recognizes the doublestranded RNA polymerase promoter results in the production of amplifiedasRNA molecules. Such asRNA molecules find utility in various downstreamapplications, including gene expression studies involving nucleic acidmicroarrays. The methods of present invention are particularly suitedfor amplification of RNA from small numbers of cells, including singlecells, which can be purified from complex cellular samples using, e.g.,micromanipulation, fluorescence-activated cell sorting (FACS) and lasermicrodissection techniques (see Player et al., Expert Rev. Mol. Diagn.4:831 (2004)).

The term “RNA polymerase recognition sequence” is intended to cover bothsingle stranded and double stranded nucleotide sequences. When in singlestranded form, the nucleotide sequence corresponds to the template ornon-template strand of a double-stranded RNA polymerase promoter.“Template strand” refers to a strand of nucleic acid on which acomplementary copy is synthesized from nucleotides or nucleotide analogsthrough the activity of a template-dependent nucleic acid polymerase.“Non-template strand” refers to the nucleic acid strand that iscomplimentary to the template strand. When in double stranded form, thenucleotide sequences correspond to both the template and non-templatestrands of a double-stranded RNA polymerase promoter.

Any method for producing sRNA molecules can be used as the source ofsuch molecules in the methods of the present invention, so long as their3′ ends comprise a nucleotide sequence corresponding to the partial 5′end of the template strand of a RNA polymerase recognition sequence. Forexample, the sRNA molecules can be produced by in vitro transcription ofcDNA molecules containing one or more RNA polymerase promoters at their3′ ends. Such methods include those disclosed in Applicants′ copendingpatent applications U.S. patent Ser. Nos. 10/979,052, 11/150,794 and11/210,602, and International Application No. PCT/US2004/014325, as wellas in U.S. patent application Ser. Nos. 10/805,171, 10/302,675,10/206,613 and 10/075,335 (each of which is specifically incorporatedherein by reference in its entirety). Commercial kits are also availablefor the production of sRNA molecules, such as, e.g., the SMART™ mRNAAmplification Kit (Clontech, Mountain View, Calif.) and the ArrayItMiniAmp mRNA Amplification Kit (ArrayIt, Sunnyvale, Calif.).

To ensure that the 3′ ends of the sRNA molecules comprise a nucleotidesequence corresponding to the partial 5′ end of the template strand of aRNA polymerase recognition sequence, the cDNA molecules are provided byreverse transcription of an RNA molecule of interest with a primerhaving a 5′ extension comprising a nucleotide sequence corresponding tothe partial 3′ end of the non-template strand of the RNA polymeraserecognition sequence. The length of the 5′ extension generally rangesfrom about 2 to about 19 nucleotides in length, preferably from about 8to about 12 nucleotides in length.

Upon reverse transcription of the RNA molecule of interest andsubsequent in vitro transcription of the resulting cDNA molecule, thenucleotide sequence of the primer extension is converted into the 3′nucleotide sequence of the sRNA molecule corresponding to the partial 5′end of the template strand of the RNA polymerase recognition sequence.Any RNA polymerase recognition sequence can be used in the methodsdescribed herein, so long as it is specifically recognized by an RNApolymerase. Preferably, the RNA polymerase recognition sequence used isrecognized by a bacteriophage RNA polymerase, such as T7, T3, or SP6 RNApolymerase. An exemplary T7 RNA polymerase recognition sequence isTAATACGACTCACTATAGGG (SEQ ID NO: 1). An exemplary T3 RNA polymeraserecognition sequence is AATTAACCCTCACTAAAGGG (SEQ ID NO: 2). Anexemplary SP6 RNA polymerase recognition sequence isAATTTAAGGTGACACTATAGAA (SEQ ID NO: 3).

For example, with reference to FIG. 1 (an embodiment previouslydescribed in Applicants′ copending U.S. patent application Ser. No.11/210,602, specifically incorporated herein by reference in itsentirety), RNA molecules (e.g., mRNA, hnRNA, rRNA, tRNA, miRNA, snoRNA,non-coding RNAs) from a source of interest are reversed transcribed intocDNA molecules using a primer having the required 5′ extension (in thecase of FIG. 1, the partial 5′ end of the template strand of the T7promoter) (see FIG. 1 a). The RNA may be obtained from any tissue orcell source, including virion, prokaryotic, and eukaryotic sources foundin any biological or environmental sample. Preferably, the source iseukaryotic tissue, more preferably mammalian tissue, even morepreferably human tissue.

Any reverse transcriptase can be used in the reverse transcriptionreaction, including thermostable and RNase H⁻ reverse transcriptases.Preferably, a RNase H⁻ reverse trancriptase is used. Numerous methodsand commercial kits for the synthesis of cDNA molecules are well knownin the art. Examples include the Superscript™ Double Strand cDNASynthesis kit (Invitrogen, Carlsbad, Calif.), the Array 50™, Array 350™and Array 900™ Detection kits (Genisphere, Hatfield, Pa.), and theCyScribe™ Post-Labelling kit (Amersham, Piscataway, N.J.).

Suitable reverse transcription primers containing the required 5′extension include single stranded oligodeoxynucleotides comprising anoligodT tail at their 3′ ends, the tail generally ranging from about 10to about 30 nucleotides in length, preferably from about 17 to about 24nucleotides in length, which anneal to RNA containing a 3′ polyA tail(e.g., mRNA). If the RNA of interest does not naturally contain a 3′polyA tail (e.g., miRNA), a polyA tail can be attached to the RNAmolecules using poly(A) polymerase (PAP) in the presence of ATP. PolyAtailing kits are commercially available and include, e.g., the Poly(A)Tailing Kit (Ambion, Austin, Tex.). Three-primer blocked RNAs can beenzymatically treated to allow tailing using, e.g., calf intestinalalkaline phosphatase or RNase 3.

Alternatively, the reverse transcription reaction can be initiated usinga random primer having the required 5′ extension, the random nucleotideportion generally ranging from about 4 to about 20 nucleotides inlength, preferably from about 6 to about 9 nucleotides in length, whichanneals to various positions along the length of each original mRNAtranscript. One of ordinary skill in the art will recognize that the useof a random primer can ultimately result in the production of sRNAmolecules that are better representative of the entire length of eachoriginal mRNA transcript than those produced using an oligodT primer.Additionally, the use of a random primer to generate cDNA in the initialsteps of the disclosed methods means that RNA that would normally beexempt from amplification, such as degraded RNA or RNA derived frombacteria, can be used to produce amplified sRNA molecules.

In some embodiments, the 3′ terminal nucleotide of the reversetranscription primer (oligodT primer, random primer, or both) is anucleotide or nucleotide analog that is not a substrate for terminaldeoxynucleotide transferase but can be extended by reversetranscriptase, such as a ribonucleotide. Such primers are not extendablewith terminal deoxynucleotidyl transferase (TdT), and thus will not betailed and amplified in the steps shown in FIGS. 1 b-1 f.

Following first strand cDNA synthesis, the resulting first round cDNAmolecules are generally purified (see FIG. 1 b). While not degrading theRNA prior to cDNA purification is preferred, cDNA that has been purifiedfollowing RNA degradation works equally well in the methods of thepresent invention. Any method that degrades RNA can be used, such astreatment with NaOH or RNase H (whether supplied in the form of a RNaseH⁺ reverse transcriptase or as a separate enzyme). Alternatively, theRNA can be left intact, with the first round cDNA molecules purifiedfrom RNA/cDNA duplexes. Numerous methods and kits exist for thepurification of DNA molecules, including, e.g., the MinElute™ PCRPurification Kit (Qiagen, Valencia, Calif.). If a reverse transcriptionprimer is used for first strand cDNA synthesis in which the 3′ terminalnucleotide is a ribonucleotide, DNA purification can be omitted. Thismay reduce sample loss and increase amplification yield, which isparticularly important when manipulating RNA from small numbers ofcells.

Following first round cDNA purification, a single strandedoligodeoxynucleotide tail is generally attached to the 3′ end of thecDNA molecules (see FIG. 1 b). The use of such oligodeoxynucleotidetails allows whole populations of nucleic acid molecules to beamplified, rather than just specific sequences. The oligodeoxynucleotidetail can be incorporated by any means that attaches deoxynucleotides toDNA. Preferably, the oligodeoxynucleotide tail is attached to the cDNAusing terminal deoxynucleotidyl transferase, or other suitable enzyme,in the presence of appropriate deoxynucleotides. Preferably, theoligodeoxynucleotide tail is a homopolymeric tail (i.e., polydA, polydG,polydC, or polydT). Preferably, the oligodeoxynucleotide tail is apolydA tail, generally ranging from about 3 to greater than 500nucleotides in length, preferably from about 20 to about 100 nucleotidesin length. Applicants have found that the use of a polydA tail reducesthe number of artifacts resulting from non-specific amplification.

Following attachment of the single stranded oligonucleotide tail to the3′ ends of the cDNA molecules, a single stranded RNA/DNA compositebridge oligonucleotide comprising a 5′ RNA portion and a 3′ DNA portionis annealed to the 3′ oligodeoxynucleotide tail (see FIG. 1 c). This isaccomplished through complementary base pairing between the 3′oligodeoxynucleotide tail and at least a portion of the 3′ DNA portionof the RNA/DNA composite bridge oligonucleotide. For example, ifoligonucleotide tail is a polydA tail, the 3′ DNA portion of the RNA/DNAcomposite bridge oligonucleotide will contain a series of thymidines atits 3′ end, generally ranging from about 3 to greater than 50nucleotides in length, preferably from about 10 to about 30 nucleotidesin length. The particular deoxynucleotide sequence of the 3′ DNA portionof the RNA/DNA composite bridge oligonucleotide does not have to beperfectly complementary to the particular nucleotide sequence of theoligodeoxynucleotide tail at the 3′ ends of the cDNA molecules, nor dotheir lengths need to match exactly, for the sequences to be consideredcomplementary to each other. Those of skill in the art will recognizethat what is required is that there be sufficient complementaritybetween the two sequences so that the RNA/DNA composite bridgeoligonucleotide can anneal to the oligodeoxynucleotide tail at the 3′end of the cDNA molecules.

In some embodiments, rather than attaching a single strandedoligodeoxynucleotide tail to the 3′ ends of the cDNA molecules, a singlestranded RNA/DNA composite bridge oligonucleotide in which the DNAportion comprises random nucleotides is annealed to the cDNA molecules.Again, the use of such a random composite bridge oligonucleotide allowswhole populations of nucleic acid molecules to be amplified, rather thanjust specific sequences. The random DNA portion of the compositeoligonucleotide generally ranges from about 3 to greater than 50nucleotides in length, preferably from about 6 to about 20 nucleotidesin length. Only those bridge oligonucleotides that hybridize to the 3′ends of the cDNA molecules will result in the synthesis of functionalRNA polymerase promoters as described below. Hybridization is preferablyperformed at about 37° C. to about 55° C., more preferably at 45° C. toabout 50° C.

In addition to the 3′ DNA portion (whether random or defined), thecomposite bridge oligonucleotide contains a 5′ RNA portion which remainssingle stranded (i.e., unannealed) following the annealing of the 3′ DNAportion of the composite bridge oligonucleotide to the 3′oligodeoxynucleotide tail. The 5′ RNA portion generally ranges fromabout 3 to greater than 50 nucleotides in length, preferably from about10 to about 30 nucleotides in length. Preferably, the particularsequence of the 5′ RNA portion is not substantially homologous to anyknown nucleic acid sequence, nor is it substantially self-complementaryor complementary to any portion of the single stranded RNA polymerasepromoter template described below.

The RNA/DNA composite bridge oligonucleotide can be blocked at its 3′end if desired, such that it is not extendable with a DNA polymerase(see FIG. 1 c). As such, the addition of reverse transcriptase with bothRNA-dependent and DNA-dependent DNA polymerase activity (e.g., MMLVreverse transcriptase, AMV reverse transcriptase, RBst DNA polymerase(Epicentre Technologies, Madison, Wis.)) and dNTPs extends the singlestranded 3′ oligonucleotide tail at the 3′ ends of the cDNA moleculessuch that the RNA portion of the bridge oligonucleotide becomes a doublestranded RNA/DNA duplex, but does not catalyze the synthesis of secondstrand cDNA (see FIG. 1 c). The RNA/DNA composite bridge oligonucleotidecan be blocked by any means that renders it incapable of being extendedwith DNA polymerase, such as by including terminal blocking groups,compounds, or moieties either attached during or after synthesis.Preferably, the RNA/DNA composite bridge oligonucleotide is blocked witha 3′ amino modifier, a 3′ deoxyterminator, or a 3′ dideoxyterminator. Asuitable blocker should not be restricted to any of those describedherein and can include any moiety that will prevent a DNA polymerasefrom extending the 3′ terminus of the RNA/DNA composite bridgeoligonucleotide.

Following extension of the 3′ oligonucleotide tail to form a RNA/DNAduplex, the RNA portion of the duplex (i.e., the RNA portion of thebridge oligonucleotide) is degraded with RNase to expose a 3′ singlestranded DNA tail on the cDNA molecules (see FIG. 1 d). Preferably, theRNase is RNase H, although other RNases, such as RNase 1 and RNase A canbe used. The RNase can be provided as part of the reverse transcriptaseor as a separate enzyme. The RNase is preferably added at substantiallythe same time as the reverse transcriptase and the bridgeoligonucleotide (see FIG. 1 c).

Following degradation of RNA portion of the RNA/DNA duplex, a singlestranded RNA polymerase promoter template is attached to the exposed 3′single stranded DNA tail on the cDNA molecules (see FIG. 1 e). This isaccomplished through complementary base pairing between the exposed 3′single stranded DNA tail and a complementary series of nucleotidespresent at the 3′ end of the single stranded promoter template,generally ranging from about 3 to greater than 50 nucleotides in length,preferably from about 10 to about 30 nucleotides in length.

The single stranded promoter template contains at its 5′ end at leastone RNA polymerase recognition sequence. The promoter template can becomposed of RNA and/or DNA, and can be blocked or unblocked at its 3′end. When composed of both RNA and DNA, the 3′ portion of the promotertemplate that hybridizes to the exposed DNA tail on the cDNA moleculesis preferably DNA, while the 5′ unhybridized portion is RNA. Forperforming multiple rounds of sRNA synthesis, the promoter templatepreferably contains at least a second different RNA polymeraserecognition sequence 3′ to the first recognition sequence (i.e., a“tandem promoter template”; see FIG. 1 c) (see Applicants' co-pendingU.S. patent application Ser. No. 11/150,794, specifically incorporatedherein by reference in its entirety). Again, any RNA polymeraserecognition sequence can be used, so long as it is specificallyrecognized by an RNA polymerase. Preferably, the RNA polymeraserecognition sequence(s) used is recognized by a bacteriophage RNApolymerase, such as T7, T3, or SP6 RNA polymerase. The RNA polymerasepromoter template is preferably added at substantially the same time asthe reverse transcriptase, bridge oligonucleotide and RNase (e.g., inthe same reaction vessel) (see FIG. 1 c), although each of the reactionscan be performed separately.

Following attachment, the reverse transcriptase from FIG. 1 c, havingDNA-dependant DNA polymerase activity, extends the exposed 3′ singlestranded DNA tail on the cDNA molecules and converts the single strandedpromoter template into one or more double stranded RNA polymerasepromoters (in the case of FIG. 1, T7 and T3 promoters) (see FIG. 1 e).Even unblocked promoter templates are not extended during the reactionbecause reverse transcriptase lacks 5′→3′ exonuclease and stranddisplacement activities. Alternatively, or in addition, to reversetranscriptase, a DNA polymerase, such as T4 DNA polymerase, T7 DNApolymerase, or Sequenase™ (USB Corporation, Cleveland, Ohio), all ofwhich lack 5′→3′ exonuclease and strand displacement activities, can beused to extend the exposed 3′ single stranded DNA tail on the cDNAmolecules and convert the single stranded promoter template into adouble stranded RNA polymerase promoter. Klenow enzyme has even beenshown in the present system to convert the promoter template into a RNApolymerase promoter without extending the template when added near theend of the reverse transcriptase/RNase promoter synthesis reaction(s)(e.g., about 5 min to about 15 min before the completion of promotersynthesis). The use of such DNA polymerases may prevent or correctincorporation errors associated with the use of reverse transcriptasealone.

To further ensure that unblocked promoter templates are not extendedduring the promoter synthesis reaction(s), a nucleotide extension can beincluded at the 3′ end of an unblocked single stranded promotertemplate. This 3′ terminal nucleotide extension, downstream of thecomplementary 3′ series of deoxynucleotides used to attach the promotertemplate to the exposed 3′ single stranded DNA tail on the cDNAmolecules, comprises a series of nucleotides identical to the 5′ end ofthe remaining DNA portion of bridge oligonucleotide, generally rangingfrom about 3 to about 10 nucleotides in length. As such, the 3′extension, which would bind to the cDNA molecules but for the presenceof the remaining DNA portion of bridge oligonucleotide, functions toprevent access to the gap or nick present between the promoter templateand the remaining DNA portion of the bridge oligonucleotide duringpromoter synthesis (see FIG. 1 e). Thus, any potential stranddisplacement during promoter synthesis is prevented as long as a DNApolymerase incapable of degrading the 3′ nucleotide extension is used inthe synthesis reactions(s) (e.g., Klenow exo⁻).

In some embodiments, rather than enzymatically synthesizing a doublestranded RNA polymerase promoter from a single stranded promotertemplate, a double stranded RNA polymerase promoter having a templatestrand and a non-template strand is attached to the 3′ ends of the firstround cDNA molecules by DNA ligation (see Applicant's co-pendingInternational Patent Application No. PCT/US2004/014325, specificallyincorporated herein by reference in its entirety). The double strandedRNA polymerase promoter contains at its 5′ end (relative to thenon-template strand) at least one RNA polymerase recognition sequence.For performing multiple rounds of sRNA synthesis, the double strandedRNA polymerase promoter preferably contains at least a second differentRNA polymerase recognition sequence 3′ to the first recognition sequence(i.e., a “tandem promoter template”) (see Applicants' co-pending U.S.patent application Ser. No. 11/150,794, specifically incorporated hereinby reference in its entirety). Attachment of the promoter is facilitatedby complementary base pairing between the exposed 3′ single stranded DNAtail on the cDNA molecules and an overhang sequence at the 3′ end of thenon-template strand of the double stranded RNA polymerase promoter thatcontains a complementary series of nucleotides, generally ranging fromabout 3 to greater than 50 nucleotides in length, preferably from about10 to about 30 nucleotides in length. Once properly positioned, thedouble stranded promoter is attached to the cDNA molecule by ligation ofthe 5′ end of the template strand of the promoter to the 3′ end of theexposed single stranded DNA tail. Any DNA ligase can be used in theligation reaction. Preferably, the DNA ligase is T4 DNA ligase.

When performing a promoter synthesis reaction (see FIG. 1 e), secondstrand cDNA can be optionally synthesized by using a random primer. Therandom primer will anneal at various positions along the first strandcDNA and be extended by any DNA-dependant DNA polymerase activity duringpromoter synthesis. The various second strand cDNA fragments can beoptionally ligated together to form a single second strand cDNAmolecule. Such second strand cDNA molecules may stabilize (i.e., removesecondary and tertiary structure) the first strand cDNA during in vitrotranscription, resulting in a higher yield of sRNA molecules.

Following synthesis or attachment of double stranded RNA polymerasepromoter, in vitro transcription is initiated by the addition ofribonucleotides and a RNA polymerase that recognizes the promoter (seeFIG. 1 f). This provides sRNA molecules having a partial RNA polymeraserecognition sequence at their 3′ ends. If a tandem promoter template wasattached to the cDNA molecules (see FIG. 1 e), in vitro transcription ispreferably initiated using a RNA polymerase that recognizes the first 5′promoter (in the case of FIG. 1, the T7 promoter) (see FIG. 1 f). Thisfacilitates second round sRNA synthesis described in further detailbelow. Methods and kits for performing in vitro transcription are wellknown in the art and include the MEGAscript™ Transcription Kit (Ambion)and the AmpliScribe™ High Yield Transcription Kits (EpicentreTechnologies).

Additional rounds of sRNA synthesis can be performed by reversetranscribing the resulting first round sRNA molecules (i.e., secondround cDNA molecules) and re-attaching or re-synthesizing a doublestranded RNA polymerase promoter onto the second-round cDNA molecules asjust described (see FIGS. 1 a-1 e), followed by a second round of invitro transcription with RNA polymerase.

If, however, a tandem promoter template was attached to the first roundcDNA molecules (see FIG. 1 e), and in vitro transcription initiatedusing a RNA polymerase that recognizes the first 5′ promoter (see FIG. 1f), additional rounds of sRNA synthesis can be performed without theneed for re-attachment or re-synthesis of the double stranded RNApolymerase promoter (see Applicants' co-pending U.S. patent applicationSer. No. 11/150,794, specifically incorporated herein by reference inits entirety).

Briefly, the first round sRNA molecules are subjected to a second roundof synthesis by first reverse transcribing the sRNA molecules into firststrand cDNA molecules (i.e. second round cDNA synthesis) using a primercomprising a nucleotide sequence complementary to the RNA polymeraserecognition sequence at the 3′ ends of the first round sRNA molecules(i.e., “corresponding” to the specific nucleotide sequence of the 5′extension of the reverse transcription primer used for first round cDNAsynthesis in the step shown in FIG. 1 a) (see FIG. 1 g). This ensuresthat the second round sRNA molecules also contain the same partial RNApolymerase recognition sequence at their 3′ ends as the first round sRNAmolecules.

Following second round cDNA synthesis, the RNA strand is degraded usingNaOH or preferably RNase H prior to optional purification of the firststrand cDNA molecules (see FIG. 1 h). Similarly, an RNase H⁺ reversetranscriptase can be used, such as MMLV.

Following RNA degradation, a single stranded promoter oligonucleotidecomplementary to the second different 3′ RNA polymerase recognitionsequence is annealed to the second round cDNA molecules throughcomplementary base pairing (see FIG. 1 h). This base pairing forms asecond RNA polymerase promoter, from which a second round of in vitrotranscription (i.e., second round sRNA molecules) is initiated by theaddition of ribonucleotides and a RNA polymerase that recognizes thesecond promoter (in the case of FIG. 1, the T3 promoter) (see FIG. 1 i).This provides second round sRNA molecules having a partial RNApolymerase recognition sequence at their 3′ ends. By incorporatingadditional different RNA polymerase recognition sequences into thepromoter template, additional rounds of sRNA synthesis can be performedas described (e.g., third round sRNA molecules, etc.). Further, by heatinactivating all enzymes between steps or before addition of RNApolymerase, using methods familiar to one skilled in the art, linear,rather than exponential, amplification can be maintained. Such linearamplification is better suited for various downstream applications, suchas gene expression studies.

In some embodiments, rather than inactivating the reverse transcriptasefollowing second round cDNA synthesis and annealing a single strandedpromoter oligonucleotide complementary to the second different RNApolymerase recognition sequence, the RNA strand is degraded using RnaseH and the tandem promoter is regenerated by the binding of excess singlestranded tandem promoter template (from the first round) to the 3′ endsof the second round cDNA molecules and the DNA-dependent DNA polymeraseactivity of the still-active reverse transcriptase (see Applicants'co-pending U.S. patent application Ser. No. 11/150,794, specificallyincorporated herein by reference in its entirety). A second round of invitro transcription can then be initiated by the addition of an RNApolymerase that recognizes either the first or second promoter. Again,the reverse transcriptase is generally heat inactivated just prior toaddition of RNA polymerase to maintain the linearity of theamplification. Those of skill in the art will recognize that the singlestranded promoter template in these embodiments need not contain two RNApolymerase recognition sequences in tandem. Rather, the promotertemplate can contain a single RNA polymerase recognition sequence, whichcan be used in place of the tandem promoter template to produce firstand second round sRNA molecules.

The sRNA molecules resulting from the above-described processes (or anyother suitable process) can then be converted into templates for thedirect synthesis of asRNA molecules by first reverse transcribing themwith a primer comprising a nucleotide sequence corresponding to at leastthe 5′ end of the non-template strand of the RNA polymerase recognitionsequence (see FIG. 2 a). The nucleotide sequence is of sufficient lengthto anneal to the nucleotide sequence at the 3′ ends of the sRNAmolecules corresponding to the partial 5′ end of the template strand ofthe RNA polymerase recognition sequence, preferably from about 2 toabout 19 nucleotides in length, preferably from about 8 to about 12nucleotides in length.

Extension of the primer with reverse transcriptase produces a doublestranded RNA/DNA duplex, the DNA portion of which now contains anucleotide sequence corresponding to the complete non-template strand ofthe RNA polymerase recognition sequence at its 5′ end (see FIG. 2 a). Atleast the 3′ portion of the RNA strand of the RNA/DNA duplex is degraded(see FIG. 2 b), thereby providing at least a partially single strandedDNA molecule having a single stranded 5′ end comprising a nucleotidesequence corresponding to the complete non-template strand of the RNApolymerase recognition sequence.

The RNA strand of the RNA/DNA duplex can be substantially completelydegraded using a RNase enzyme separate from reverse transcriptase, suchas RNase H (see FIG. 2 b). Addition of an exogenous primer in thepresence of a DNA polymerase produces a double strand stranded DNAmolecule such that the nucleotide sequence at the 5′ end of the firstDNA strand corresponding to the complete non-template strand of said RNApolymerase recognition sequence is converted into a double stranded RNApolymerase promoter (see FIG. 2 c).

The exogenous primer will comprise a nucleotide sequence of sufficientcomplementarity such that it anneals to the first DNA strand, preferablyto the 3′ region of the first DNA strand. More preferably, the exogenousprimer anneals to the 3′ most nucleotides of the first DNA strand. Thisensures that most or all of the information present in each sRNAmolecule or a particular sRNA molecule is captured during second strandDNA synthesis.

The exogenous primer can comprise any nucleotide sequence as long as itis complementary to a sequence of the first DNA strand known to theend-user. This sequence can either be naturally occurring or can beengineered by processes such as, e.g., those described above. Forexample, if an RNA/DNA composite bridge oligonucleotide was used asshown in FIG. 1 c, the exogenous primer can comprise a nucleotidesequence corresponding to the complement of at least a portion of theDNA portion of the bridge oligonucleotide, as shown in FIG. 2 c. If apolydA tail was attached to the cDNA molecules as shown in FIG. 1 b (orin a similar method), the exogenous primer can comprise a series ofthymidines of sufficient length to anneal to the first DNA strand in thestep shown in FIG. 2 c. Alternatively, the exogenous primer can comprisea naturally occurring gene-specific sequence, such that gene-specificasRNA molecules can be produced by in vitro transcription in the stepshown in FIG. 2 d.

In the in vitro transcription reaction, addition of ribonucleotides anda RNA polymerase that recognizes the promoter formed in the step shownin FIG. 2 c results in the synthesis of asRNA molecules directly fromsRNA molecules (see FIG. 2 d), thereby providing an additional round ofRNA amplification.

Alternatively, the RNA strand of the RNA/DNA duplex can be partiallydegraded using a reverse transcriptase having RNAse activity, such as,e.g., AMV reverse transcriptase or MMLV RNase H⁺, without the additionof a separate RNase enzyme. In this one-step embodiment, the RNaseactivity of the reverse transcriptase degrades the RNA template strandessentially while synthesizing the complementary cDNA strand, therebyleaving a remaining portion of the RNA strand which can serve as aprimer for second strand DNA synthesis. Again, addition ofribonucleotides and a RNA polymerase that recognizes the resultingpromoter results in the synthesis of asRNA molecules directly from sRNAmolecules, providing an additional round of RNA amplification.

The resulting asRNA molecules may represent up to a billion-foldamplification of each target RNA and can be used directly in geneexpression studies. For example, the in vitro transcription reaction(see FIG. 2 d) can be performed in the presence of detectably labelednucleotides, such as fluorescently labeled nucleotides, to producedirectly labeled asRNA molecules. Such nucleotides include nucleotideslabeled with Cy3 and Cy5.

In alternative embodiments, the asRNA molecules are labeled indirectlyfollowing their synthesis. For example, in vitro transcription reactioncan be performed in the presence amino allyl nucleotides (e.g., aminoallyl UTP), followed by coupling to a NHS ester label (e.g., biotin, Cydye).

In some embodiments, labeled cDNA molecules are synthesized rather thanasRNA molecules. For example, the reverse transcription reaction shownin FIG. 2 a can be performed in the presence of detectably labelednucleotides, such as fluorescently labeled nucleotides (e.g., Cy3 andCy5 labeled nucleotides), to produce directly labeled cDNA molecules, orthe reaction can be performed in the presence amino allyl nucleotides(e.g., amino allyl UTP), followed by coupling to a NHS ester label(e.g., biotin, Cy dye), to produce indirectly labeled cDNA molecules.

Alternatively, the reverse transcription primer used in FIG. 2 a cancomprise a capture nucleotide sequence at its 5′ end in addition to thenucleotide sequence necessary to anneal to the nucleotide sequence atthe 3′ ends of the sRNA molecules. Any defined nucleotide sequence canfunction as the capture nucleotide sequence, generally ranging fromabout 10 to about 100 nucleotides in length, preferably from about 25 toabout 35 nucleotides in length. Preferably, the capture nucleotidesequence shares no significant homology with any known gene sequence.

The capture nucleotide sequence becomes incorporated into the cDNAmolecules during the reverse transcription reaction shown in FIG. 2 a.The resulting capture nucleotide sequence-containing cDNA molecules canbe labeled indirectly using, e.g., 3DNA™ dendrimer technology(Genisphere, Hatfield, Pa.). The surfaces of these dendrimers comprisemultiple nucleotide sequences complementary to the capture nucleotidesequence, as well as multiple attachment sites for labeled (e.g., Cydye) oligonucleotides. Such dendritic nucleic acid reagents are furtherdescribed in Nilsen et al., J. Theor. Biol., 187:273 (1997); Stears etal., Physiol. Genomics, 3:93 (2000); and U.S. Pat. Nos. 5,175,270;5,484,904; 5,487,973; 6,072,043; 6,110,687; and 6,117,631, each of whichis specifically incorporated herein by reference in its entirety.

The labeled asRNA and cDNA molecules are useful as reagents for geneexpression studies. The labeled molecules can be annealed to a nucleicacid microarray containing complementary polynucleotides (e.g., probes).As used herein, “microarray” is intended to include any solid supportcontaining nucleic acid probes, including slides, chips, membranes,beads, and microtiter plates. Examples of commercially availablemicroarrays include the GeneChip® microarray (Affymetrix, Santa Clara,Calif.), CodeLink™ microarray (Amersham Biosciences, Piscataway, N.J.),Agilent (Palo Alto, Calif.) Oligo microarray, and OciChip™ microarray(Ocimum Biosolutions, Indianapolis, Ind.). If the asRNA and cDNAmolecules are labeled indirectly, they can be labeled either prior to orfollowing microarray hybridization.

The methods and compositions of the present invention can beconveniently packaged in kit form. Such kits can be used in variousresearch and diagnostic applications. For example, methods and kits ofthe present invention can be used to facilitate a comparative analysisof expression of one or more genes in different cells or tissues,different subpopulations of the same cells or tissues, differentphysiological states of the same cells or tissue, differentdevelopmental stages of the same cells or tissue, or different cellpopulations of the same tissue. Such analyses can reveal statisticallysignificant differences in the levels of gene expression, which,depending on the cells or tissues analyzed, can then be used tofacilitate diagnosis of various disease states.

A wide variety of kits may be prepared according to present invention.For example, a kit may include one or more primers comprising anucleotide sequence corresponding to at least a portion of the 5′ end ofthe non-template strand of a RNA polymerase recognition sequence; andinstructional materials for synthesizing asRNA molecules directly fromsRNA molecules using said primer.

In addition, or alternatively, the kit can include reagents andinstructional materials for synthesizing sRNA molecules from which asRNAmolecules can be directly synthesized. For example, the kit may includeone or more reverse transcription primers having a 5′ extensioncomprising a nucleotide sequence corresponding to the partial 3′ end ofthe non-template strand of a RNA polymerase recognition sequence; asingle stranded promoter template comprising at least one RNA polymeraserecognition sequence; a single stranded RNA/DNA composite bridgeoligonucleotide comprising a RNA sequence 5′ of a DNA sequence; andinstructional materials for synthesizing sRNA molecules from which asRNAmolecules can be directly synthesized.

For performing additional rounds of sRNA synthesis, the kit can furtherinclude one or more second round reverse transcription primerscomprising a nucleotide sequence corresponding to the nucleotidesequence of the 5′ extension of a first round reverse transcriptionprimer or primers; and a single stranded promoter oligonucleotidecomplementary to a second RNA polymerase recognition sequence of thepromoter template and the appropriate instructional materials.

While the instructional materials typically comprise written or printedmaterials, they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

The kits of the present invention may further include one or more of thefollowing components or reagents: one or more reverse transcriptases(RNase H⁺ and/or RNase H⁻); an RNase inhibitor (e.g., Superase-In™); anRNase (e.g., RNase H); an enzyme for attaching an oligodeoxynucleotidetail onto the 3′ ends of single stranded cDNA molecules (e.g., terminaldeoxynucleotidyl transferase); an enzyme for attaching anoligoribonucleotide tail onto the 3′ ends of RNA molecules (e.g.,poly(A) polymerase); one or more DNA-dependent DNA polymerases (e.g.,Klenow enzyme); an enzyme for ligating a double stranded RNA polymerasepromoter onto the 3′ ends of single stranded DNA molecules (e.g., T4 DNAligase); one or more RNA-dependent RNA polymerases (e.g., T7 polymerase,T3 polymerase, SP6 polymerase); one or primers comprising a capturenucleotide sequence; one or more second strand DNA synthesis primers;dNTP mix (e.g., dATP, dCTP, dGTP, dTTP); dATP; NTP mix (e.g., ATP, CTP,GTP, UTP); low UTP NTP mix; labeled nucleotides; reaction buffers;salts; nuclease-free water; and/or containers, vials, reaction tubes,and the like compatible with the synthesis of asRNA molecules directlyfrom sRNA molecules according to the methods of the present invention.The components and reagents may be provided in containers with suitablestorage media.

Specific embodiments according the present invention will now bedescribed in the following examples. The examples are illustrative only,and are not intended to limit the remainder of the disclosure in anyway.

EXAMPLES Example 1

One Round of sRNA Synthesis Followed by asRNA Synthesis

1. First Strand cDNA Synthesis

For each RNA sample, purified using the RNAqueous® Kit (Ambion), thefollowing RNA/primer mix was prepared on ice:

-   -   1-8 μl total RNA (not exceeding 2 ng)    -   2 μl first round oligodT sequence specific RT primer (50 ng/μl)        (5′-CTC ACT ATA GGG TTT TTT TTT TTT TTT TTT V-3′, where V=C, G        or A deoxyribonucleotides; SEQ ID NO: 4)    -   1 μl first round random sequence specific RT primer (2× by mass        of RNA) (5′-CTC ACT ATA GGG NNN NNN NNN-3, where N=A, G, C or T        deoxyribonucleotides at random; SEQ ID NO: 5)    -   RNase-free water to 11 μl

The first round RT primers have a 5′ extension comprising a nucleotidesequence corresponding to the partial 3′ end of the non-template strandof the T7 promoter. The RNA/primer mixture was heated at 80° C. for 10minutes and immediately cooled on ice for 1-2 min. The mixture was thenmixed with 9 μl of a Master Mixture solution to bring the final volumeto 20 μl containing 1× RT buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3mM MgCl₂), 10 mM dithiothreitol (DTT), 0.5 mM each dNTP, 10 USuperase-In™ (Ambion), and 200 U Superscript™ II reverse transcriptase(Invitrogen). The mixture was briefly centrifuged and incubated at 42°C. for 2 hrs. Following a brief centrifugation, the reaction wasadjusted to 100 μl with 1× TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

2. cDNA Purification

The reaction was purified using the MinElute™ PCR Purification Kit(Qiagen) according to the manufacturer's protocol. Briefly, the cDNAreaction was adjusted to 600 μl with PB buffer provided by themanufacturer. The cDNA reaction was applied to the MinElute™ column andmicrofuged for 1 minute. The flow-through in the collection tube wasdiscarded, and the column washed with 750 μl PE buffer provided by themanufacturer. The flow-through in the collection tube was discarded, andthe column washed with 500 μl 80% ethanol. The flow-through in thecollection tube was discarded, and the column microfuged with the capopen for 5 minutes to dry the resin. The column was placed in a clean1.5 ml microfuge tube, and the column membrane incubated with 10 μl EBbuffer provided by the manufacturer for 2 minutes at room temperature.The first strand cDNA molecules were eluted by microfugation for 2minutes.

3. Tailing of First Strand cDNA

The first strand cDNA molecules were heated at 80° C. for 10 minutes andimmediately cooled on ice for 1-2 min. The cDNA molecules in 10 μl werethen mixed with 10 μl of a Master Mixture solution to bring the finalvolume to 20 μl containing 1× Tailing buffer (10 mM Tris-HCl, pH 7.0, 10mM MgCl₂), 0.04 mM dATP, and 15 U terminal deoxynucleotidyl transferase(Roche Diagnostics, Indianapolis Ind.). The mixture was brieflycentrifuged and incubated at 37° C. for 2 min. The reaction was stoppedby heating at 80° C. for 10 min and cooled at room temperature for 1-2minutes.4. T7/T3 Promoter Synthesis

One μl of T7/T3 RNA polymerase promoter template oligonucleotide (5′-TAATAC GAC TCA CTA TAG GGA GAA ATT AAC CCT CAC T-3′; SEQ ID NO: 6) (100ng/μl) and 1 μl of RNA/DNA composite bridge oligonucleotide (5′-rGrArArArUrU rArArC rCrCrU rCrArC rUAA AGG GAT TTT TTT TTT TTT T-3′; SEQ IDNO: 7) (100 ng/μl) containing a 3′ amino modifier was added to theoligodA-tailed cDNA molecules and the mixture incubated at 37° C. for 10min to anneal the strands. The T7/T3 RNA polymerase promoter templatecontains a T7 RNA polymerase promoter template 5′ to a T3 RNA polymeraserecognition sequence. The tailed cDNA molecules/bridgeoligonucleotide/promoter template mixture was then mixed with 3 μl of aMaster Mixture solution to bring the final volume to 25 μl containing 1×Polymerase buffer (10 mM Tris-HCl, pH 7.0, 10 mM MgCl₂), 0.4 mM eachdNTP, 200 U Superscript II reverse transcriptase (Invitrogen) and 2 URNase H (Invitrogen). The mixture was briefly centrifuged and incubatedat 37° C. for 30 minutes. The reaction was stopped by heating at 65° C.for 15 min and placed on ice.

5. T7 In Vitro Sense Transcription

One-half of the promoter synthesis reaction (12.5 μl) was heated at 37°C. for 10-15 min to re-anneal the T7T3 promoter strands and then mixedwith 12.5 μl of a Master Mixture solution to bring the final volume to25 μl containing 1× Reaction buffer, 7.5 mM each rNTP, and 2 μl T7 RNApolymerase (MEGAscrip™ Transcription Kit, Ambion). The mixture wasbriefly centrifuged and incubated in a thermocycler with a heated lid at37° C. for 4-16 hrs. Alternatively, the mixture was incubated in a 37°C. heat block for 15 min, followed by incubation in an air hybridizationoven at 37° for 4-16 hrs. It is essential to avoid evaporation andcondensation of the reaction during this step.

6. Reverse Transcription of First Round sRNA

Fifty ng of sRNA in a volume of 4 μl was mixed with 2 μl of antisense RTprimer (100 ng/μl) (5′-CAT TAA TAC GAC TCA CTA TAG G-3′; SEQ ID NO: 8)and heated at 80° C. for 10 min. The antisense RT primer comprises anucleotide sequence corresponding to the non-template strand of the T7promoter and is therefore of sufficient length to anneal to thenucleotide sequence at the 3′ ends of the sRNA molecules. The reactionwas immediately iced for 2 min, briefly centrifuged, and returned toice. The mixture was then mixed with 4 μl of a Master Mixture solutionto bring the final volume to 10 μl containing 1× RT buffer (50 mMTris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂), 10 mM dithiothreitol (DTT),0.5 mM each dNTP, 10 U Superase-In™ (Ambion), and 200 U MMLV reversetranscriptase (RNase H⁺) (Promega). The mixture was briefly centrifugedand incubated at 42° C. for 2 hrs. The reaction was stopped by heatingat 80° C. for 10 min and cooled at room temperature for 1-2 minutes.

T7 In Vitro Antisense Transcription

The reverse transcription reaction was heated to 37° C. for 10-15 minand then mixed with 14.5 μl of a Master Mixture solution to bring thefinal volume to 24.5 μl containing 1× Reaction buffer, 7.5 mM each rNTP,and 2 μl T7 RNA polymerase (MEGAscript™ Transcription Kit, Ambion). Themixture was briefly centrifuged and incubated in a thermocycler with aheated lid at 37° C. for 4-16 hrs. Alternatively, the mixture wasincubated in a 37° C. heat block for 15 min, followed by incubation inan air hybridization oven at 37° for 4-16 hrs. It is essential to avoidevaporation and condensation of the reaction during this step.

Replicate amplifications were performed starting with 50 ng of sRNA orwater alone (negative control). On average, 25-30 μg of amplified asRNAwas recovered after amplifying 50 ng of sRNA vs. 0.5-1 μg ofnon-specific amplification product when using only water in the reversetranscription reaction. Sense and antisense RNAs were run on a 1%agarose gel to visualize the product size (see FIG. 3, lanes 2 and 3).Generally, the products ranged in size from about 0.1 to 4 kb, whichreflects the normal size distribution of mRNA.

Example 2

Two Rounds of sRNA Synthesis Followed by asRNA Synthesis

1. First Strand cDNA Synthesis

For each RNA sample, purified using the RNAqueous® Kit (Ambion), thefollowing RNA/primer mix was prepared on ice:

-   -   1-8 μl total RNA (not exceeding 2 ng)    -   2 μl first round oligodT sequence specific RT primer (50 ng/pl)        (5′-CTC ACT ATA GGG TTT TTT TTT TTT TTT TTT V-3′, where V=C, G        or A deoxyribonucleotides; SEQ ID NO: 4)    -   1 μl first round random sequence specific RT primer (2× by mass        of RNA) (5′-CTC ACT ATA GGG NNN NNN NNN-3, where N=A, G, C or T        deoxyribonucleotides at random; SEQ ID NO: 5)        RNase-free water to 11 μl

The first round RT primers have a 5′ extension comprising a nucleotidesequence corresponding to the partial 3′ end of the non-template strandof the T7 promoter. The RNA/primer mixture was heated at 80° C. for 10minutes and immediately cooled on ice for 1-2 min. The mixture was thenmixed with 9 μl of a Master Mixture solution to bring the final volumeto 20 μl containing 1× RT buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3mM MgCl₂), 10 mM dithiothreitol (DTT), 0.5 mM each dNTP, 10 USuperase-In™ (Ambion), and 200 U Superscript™ II reverse transcriptase(Invitrogen). The mixture was briefly centrifuged and incubated at 42°C. for 2 hrs. Following a brief centrifugation, the reaction wasadjusted to 100 μl with 1× TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

2. cDNA Purification

The reaction was purified using the MinElute™ PCR Purification Kit(Qiagen) according to the manufacturer's protocol. Briefly, the cDNAreaction was adjusted to 600 μl with PB buffer provided by themanufacturer. The cDNA reaction was applied to the MinElute™ column andmicrofuged for 1 minute. The flow-through in the collection tube wasdiscarded, and the column washed with 750 μl PE buffer provided by themanufacturer. The flow-through in the collection tube was discarded, andthe column washed with 500 μl 80% ethanol. The flow-through in thecollection tube was discarded, and the column microfuged with the capopen for 5 minutes to dry the resin. The column was placed in a clean1.5 ml microfuge tube, and the column membrane incubated with 10 μl EBbuffer provided by the manufacturer for 2 minutes at room temperature.The first strand cDNA molecules were eluted by microfugation for 2minutes.

3. Tailing of First Strand cDNA

The first strand cDNA molecules were heated at 80° C. for 10 minutes andimmediately cooled on ice for 1-2 min. The cDNA molecules in 10 μl werethen mixed with 10 μl of a Master Mixture solution to bring the finalvolume to 20 μl containing 1× Tailing buffer (10 mM Tris-HCl, pH 7.0, 10mM MgCl₂), 0.04 mM dATP, and 15 U terminal deoxynucleotidyl transferase(Roche Diagnostics). The mixture was briefly centrifuged and incubatedat 37° C. for 2 min. The reaction was stopped by heating at 80° C. for10 min and cooled at room temperature for 1-2 minutes.

4. T7/T3 Promoter Synthesis

One μl of T7/T3 RNA polymerase promoter template oligonucleotide (5′-TAATAC GAC TCA CTA TAG GGA GAA ATT AAC CCT CAC T-3′; SEQ ID NO: 6) (100ng/μl) and 1 μl of RNA/DNA composite bridge oligonucleotide (5′-rGrArArArUrU rArArC rCrCrU rCrArC rUAA AGG GAT TTT TTT TTT TTT T-3′; SEQ IDNO: 7) min, briefly centrifuged, and returned to ice. One μl dNTP mix(10 mM each) and 1 μl Superscript™ II reverse transcriptase (200 U/μl;Invitrogen) was added, and the RT reaction incubated at 42° C. for 1 hr.One μl RNase H (2. U/μl) (Invitrogen) was added, and the reactionincubated at 37° C. for 20 min. The reaction was then incubated at 65°C. to stop enzyme activity.

7. T3 Promoter Formation

Two μl of T3 promoter oligonucleotide (50 ng/μl) (5′-GAA ATT AAC CCT CACTAA AGG G-3′; SEQ ID NO: 10) was added to the second round cDNAreaction. The T3 oligonucleotide is complementary to the T3 RNApolymerase recognition sequence of the initial T7/T3 RNA polymerasepromoter template. The reaction was incubated at 37° for 10 min toanneal the strands.

8. T3 In Vitro Sense Transcription

The T3 promoter synthesis reaction was mixed with 19 μl of a MasterMixture solution to bring the final volume to 25 μl containing 1×Reaction buffer, 7.5 mM each rNTP, and 2 μl T3 RNA polymerase(MEGAscript™ Transcription Kit, Ambion). The mixture was brieflycentrifuged and incubated in a thermocycler with a heated lid at 37° C.for 4-16 hrs. Alternatively, the mixture was incubated in a 37° C. heatblock for 15 min, followed by incubation in an air hybridization oven at37° for 4-16 hrs. It is essential to avoid evaporation and condensationof the reaction during this step.

9. sRNA Purification and Quantitation

The second round sRNA molecules were purified using the RNeasy Kit(Qiagen) following manufacturer's protocol for RNA cleanup. The purifiedsRNA molecules were eluted twice in 50 μl RNase-free water andquantified by UV-spectrophotometry in 0.1× TE Buffer, pH 8.0 at awavelength ratio of 260/280.

10. Reverse Transcription of Second Round sRNA

Fifty ng of second round SRNA in a volume of 4 μl was mixed with 2 μl ofantisense RT primer (100 ng/μl) (5′-CAT TAA TAC GAC TCA CTA TAG G-3′;SEQ ID NO: 8) and heated at 80° C. for 10 min. The antisense RT primercomprises a nucleotide sequence corresponding to the non-template strandof the T7 promoter and is therefore of sufficient length to anneal tothe nucleotide sequence at the 3′ ends of the second round sRNAmolecules. The reaction was immediately iced for 2 min, brieflycentrifuged, and returned to ice. The mixture was then mixed with 4 μlof a Master Mixture solution to bring the final volume to 10 μlcontaining 1× RT buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mMMgCl₂), 10 mM dithiothreitol (DTT), 0.5 mM each dNTP, 10 U Superase-In™(Ambion), and 200 U MMLV reverse transcriptase (RNase H+) (Promega). Themixture was briefly centrifuged and incubated at 42° C. for 2 hrs. Thereaction was stopped by heating at 80° C. for 10 min and cooled at roomtemperature for 1-2 minutes.

11. T7 In Vitro Antisense Transcription

The reverse transcription reaction heated to 37° C. for 10-15 min andthen mixed with 14.5 μl of a Master Mixture solution to bring the finalvolume to 24.5 μl containing 1× Reaction buffer, 7.5 mM each rNTP, and 2μl T7 RNA polymerase (MEGAscript™ Transcription Kit, Ambion). Themixture was briefly centrifuged and incubated in a thermocycler with aheated lid at 37° C. for 4-16 hrs. Alternatively, the mixture wasincubated in a 37° C. heat block for 15 min, followed by incubation inan air hybridization oven at 37° for 4-16 hrs. It is essential to avoidevaporation and condensation of the reaction during this step.

Replicate amplifications were performed starting with 50 ng of sRNA orwater alone (negative control). On average, 25-30 μg of amplified asRNAwas recovered after amplifying 50 ng of sRNA vs. 0.5-1 μg ofnon-specific amplification product when using only water in the reversetranscription reaction. Sense and antisense RNAs were run on a 1%agarose gel to visualize the product size (see FIG. 3, lanes 4 and 5).Generally, the products ranged in size from about 0.1 to 4 kb, whichreflects the normal size distribution of mRNA. This size range wasmaintained even after two rounds of sRNA synthesis.

Example 3

A kit for performing one or more rounds of sRNA synthesis followed byasRNA synthesis was assembled with the following components:

-   -   First round oligodT reverse transcription primer having a 5′        extension comprising a nucleotide sequence corresponding to the        partial 3′ end of the non-template strand of the T7 promoter (50        ng/μl);    -   First round random reverse transcription primer having a 5′        extension comprising a nucleotide sequence corresponding to the        partial 3′ end of the non-template strand of the T7 promoter        (250 ng/μl);    -   5× reverse transcription buffer (250 mM Tris-HCl, pH 8.3, 375 mM        KCl, 15 MM MgCl₂, 0.1 M DTT);    -   dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP);    -   Superase-In™ RNase Inhibitor (Ambion);    -   0.2 mM dATP;    -   10× tailing buffer (100 mM Tris-HCl, pH 7.0, 100 mM MgCl₂);    -   Terminal deoxynucleotidyl transferase (7.5 U/μl);    -   RNA/DNA composite bridge oligonucleotide (100 ng/μl);    -   T7/T3 RNA polymerase promoter template (50 ng/μl);    -   NTP mix (ATP, GTP, CTP, and UTP) (75 mM each);    -   Low UTP NTP mix (75 mM ATP, 75 mM GTP, 75 mM CTP, and 25 mM        UTP);    -   10× RNA polymerase reaction buffer (Ambion);    -   Second round reverse transcription primer comprising a        nucleotide sequence corresponding to the specific nucleotide        sequence of the 5′ extension of the first round reverse        transcription primers (50 ng/μl);    -   T7 promoter oligonucleotide (50 ng/μl);    -   T3 promoter oligonucleotide (50 ng/μl);    -   T7 enzyme mix (Ambion);    -   T3 enzyme mix (Ambion);    -   RNase H (2 U/μl);    -   Antisense reverse transcription primer comprising a nucleotide        sequence corresponding to the non-template strand of the T7        promoter (50 ng/μl);    -   MMLV reverse transcriptase RNase H⁺ (Invitrogen); and    -   Nuclease-free water.

The components were placed in numbered vials and placed in a containerwith a printed instruction manual for multiple rounds of sRNA synthesisusing the kit components.

All publications cited in the specification, both patent publicationsand non-patent publications, are indicative of the level of skill ofthose skilled in the art to which this invention pertains. All thesepublications are herein fully incorporated by reference to the sameextent as if each individual publication were specifically andindividually indicated as being incorporated by reference.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the following claims.

1. A method for synthesizing at least one asRNA molecule directly fromat least one sRNA molecule, comprising: a) providing at least one sRNAmolecule having a 5′ end and a 3′ end, said 3′ end comprising a firstnucleotide sequence corresponding to the partial 5′ end of the templatestrand of a RNA polymerase recognition sequence; b) annealing to the 3′end of said sRNA molecule a primer having a 5′ end and a 3′ end, saidprimer comprising a second nucleotide sequence corresponding to at the5′ end of the non-template strand of said RNA polymerase recognitionsequence sufficient in length to anneal to said first nucleotidesequence corresponding to the partial 5′ end of the template strand ofsaid RNA polymerase recognition sequence; c) extending the 3′ end ofsaid primer such that a double stranded RNA/DNA duplex is formed; d)degrading at least the 3′ portion of the RNA strand of said doublestranded RNA/DNA duplex, thereby providing at least a partially singlestranded DNA molecule having a single stranded 5′ end, said 5′ endcomprising a third nucleotide sequence corresponding to the completenon-template strand of said RNA polymerase recognition sequence; e)synthesizing at least a partially double stranded DNA molecule from saidat least partially single stranded DNA molecule such that said thirdnucleotide sequence corresponding to the complete non-template strand ofsaid RNA polymerase recognition sequence is converted into a doublestranded RNA polymerase promoter; and f) initiating RNA transcriptionusing an RNA polymerase which recognizes said double stranded RNApolymerase promoter, thereby synthesizing at least one asRNA moleculedirectly from at least one sRNA molecule.
 2. The method of claim 1,wherein in step e) the double stranded RNA polymerase promoter is abacteriophage promoter.
 3. The method of claim 1, wherein in step d) theRNA strand of the double stranded RNA/DNA duplex is substantiallycompletely degraded using a RNase enzyme separate from reversetranscriptase.
 4. The method of claim 3, wherein step e) comprisesaddition of an exogenous primer.
 5. The method of claim 4, the exogenousprimer comprises a nucleotide sequence of sufficient complementaritysuch that it anneals to the at least partially single stranded DNAmolecule.
 6. The method of claim 5, wherein the exogenous primer annealsto the 3′ most nucleotides of the at least partially single stranded DNAmolecule.
 7. The method of claim 5, wherein the exogenous primer iscomplementary to a complementary to naturally occurring gene-specificsequence on the at least partially single stranded DNA molecule.
 8. Themethod of claim 5, wherein the exogenous primer is complementary to acomplementary to an engineered sequence on the at least partially singlestranded DNA molecule.
 9. The method of claim 1, wherein in step d) theRNA strand of the double stranded RNA/DNA duplex is partially degradedusing a reverse transcriptase having RNAse activity without the additionof a separate RNase enzyme.
 10. The method of claim 9, wherein in stepe) a remaining portion of the RNA strand serves as a primer for secondstrand DNA synthesis.
 11. The method of claim 1, wherein step f)comprises initiating RNA transcription in the presence of fluorescentlylabeled nucleotides.
 12. The method of claim 1, wherein step f)comprises RNA transcription initiating in the presence of amino allylnucleotides.
 13. The method of claim 1, wherein step a) comprises: g)providing at least one RNA molecule having a 5′ end and a 3′ end; andsynthesizing at least one single stranded cDNA molecule from said RNAmolecule or molecules using a primer having a 5′ extension comprising afourth nucleotide sequence corresponding to the partial 3′ end of thenon-template strand of the RNA polymerase recognition sequence.
 14. Themethod of claim 13, wherein the primer is a random primer, an oligodTprimer or a combination thereof.
 15. The method of claim 13, furthercomprising: (h) attaching an oligodeoxynucleotide tail having a 5′ endand 3′ end onto the 3′ end of the cDNA molecule or molecules; (i)annealing to said oligodeoxynucleotide tail a single stranded RNA/DNAcomposite bridge oligonucleotide comprising a 5′ RNA portion and a 3′DNA sequence portion, such that the RNA portion remains single stranded;(j) extending the 3′ end of said oligodeoxynucleotide tail, such thatsaid single stranded RNA portion becomes a double stranded RNA/DNAduplex; (k) degrading the RNA portion of said RNA/DNA duplex, therebyexposing a 3′ single stranded DNA tail; (l) annealing to said 3′ singlestranded DNA tail a single stranded promoter template comprising atleast one RNA polymerase recognition sequence; (m) extending said 3′single stranded DNA tail such that said at least one single stranded RNApolymerase recognition sequence is converted into at least one RNApolymerase promoter; (n) and initiating RNA transcription using a RNApolymerase which recognizes said at least one RNA polymerase promoter,thereby providing at least one sRNA molecule having a 5′ end and a 3′end, said 3′ end comprising a first nucleotide sequence corresponding tothe partial 5′ end of the template strand of a RNA polymeraserecognition sequence.
 16. The method of claim 15, wherein steps l)through m) are performed substantially at the same time.
 17. The methodof claim 15, wherein in step l) the single stranded promoter templatecomprises a first RNA polymerase recognition sequence and a second RNApolymerase recognition sequence 3′ to said first recognition sequence,wherein said first and second RNA polymerase recognition sequence aredifferent.
 18. The method of claim 17, wherein the first and second RNApolymerase recognition sequences are bacteriophage RNA polymeraserecognition sequences.
 19. The method of claim 17, wherein in step n)RNA transcription is initiated using a RNA polymerase which recognizessaid the first RNA polymerase recognition sequence.
 20. The method ofclaim 19, further comprising: (o) synthesizing at least one cDNAmolecule having a 5′ end and 3′ end from the sRNA molecule or moleculesusing a primer having a nucleotide sequence corresponding to the fourthnucleotide sequence of the 5′ extension of the primer in step g),thereby forming a double stranded sRNA/cDNA duplex; (p) degrading thesRNA portion of said sRNA/cDNA duplex, thereby providing a singlestranded cDNA molecule; (q) annealing to said single stranded cDNAmolecule a single stranded promoter oligonucleotide complementary to thesecond different RNA polymerase recognition sequence such that a seconddifferent RNA polymerase promoter is formed; and (r) initiating RNAtranscription using an RNA polymerase which recognizes said seconddifferent RNA polymerase promoter, thereby providing at least one sRNAmolecule having a 5′ end and a 3′ end, said 3′ end comprising a firstnucleotide sequence corresponding to the partial 5′ end of the templatestrand of a RNA polymerase recognition sequence.
 21. The method of claim20, wherein the first and second RNA polymerase recognition sequencesare bacteriophage RNA polymerase recognition sequences.
 22. A method forsynthesizing at least one cDNA molecule directly from at least one sRNAmolecule, comprising: a) providing at least one sRNA molecule having a5′ end and a 3′ end, said 3′ end comprising a first nucleotide sequencecorresponding to the partial 5′ end of the template strand of a RNApolymerase recognition sequence; b) annealing to the 3′ end of said sRNAmolecule a primer having a 5′ end and a 3′ end, said primer comprising asecond nucleotide sequence corresponding to at the 5′ end of thenon-template strand of said RNA polymerase recognition sequencesufficient in length to anneal to said first nucleotide sequencecorresponding to the partial 5′ end of the template strand of said RNApolymerase recognition sequence; and c) extending the 3′ end of saidprimer with reverse transcriptase, thereby synthesizing at least onecDNA molecule directly from at least one sRNA molecule.
 23. The methodof claim 22, wherein step c) comprises extending the 3′ end of theprimer in the presence of detectably labeled nucleotides.
 24. The methodof claim 23, wherein the detectable labeled nucleotides arefluorescently labeled nucleotides.
 25. The method of claim 23, whereinthe detectable labeled nucleotides are amino allyl nucleotides.
 26. Themethod of claim 22, wherein the primer in step b) further comprises acapture nucleotide sequence at its 5′ end.
 27. The method of claim 26,further comprising indirectly labeling the cDNA molecule or moleculeswith a molecule comprising a nucleotide sequence complementary to thecapture nucleotide sequence.
 28. The method of claim 27, wherein themolecule is a nucleic acid dendrimer.
 29. A method for probing a nucleicacid microarray, comprising: contacting a nucleic acid microarray withat least one asRNA molecule synthesized by the method of claim
 1. 30. Amethod for probing a nucleic acid microarray, comprising: contacting anucleic acid microarray with at least one cDNA molecule synthesized bythe method of claim
 22. 31. A kit for synthesizing asRNA moleculesand/or cDNA molecules directly from a sRNA molecule, comprising: one ormore first primers comprising a nucleotide sequence corresponding to atleast a portion of the 5′ end of the non-template strand of a RNApolymerase recognition sequence; and instructional materials forsynthesizing asRNA and/or molecules directly from sRNA molecules usingsaid first primer.
 32. The kit of claim 31, further comprising one ormore reagents and instructional materials for synthesizing sRNAmolecules from which asRNA and/or cDNA molecules can be directlysynthesized.
 33. The kit of claim, 32 comprising: one or more secondprimers having a 5′ extension comprising a nucleotide sequencecorresponding to the partial 3′ end of the non-template strand of a RNApolymerase recognition sequence.
 34. The kit of claim 33, furthercomprising a single stranded promoter template comprising at least oneRNA polymerase recognition sequence; and a single stranded RNA/DNAcomposite bridge oligonucleotide comprising a RNA sequence 5′ of a DNAsequence.
 35. The kit of claim 34, further comprising one or more thirdprimers comprising a nucleotide sequence corresponding to the nucleotidesequence of the 5′ extension of the second primer or primers; and asingle stranded promoter oligonucleotide complementary to a second RNApolymerase recognition sequence of the promoter template.